GB2579856A

GB2579856A - MHC Class I associated peptides for prevention and treatment of hepatitis B virus infection

Info

Publication number: GB2579856A
Application number: GB1820614.4A
Authority: GB
Inventors: Philip Ramila
Original assignee: Emergex Vaccines Holding Ltd
Current assignee: Emergex Vaccines Holding Ltd
Priority date: 2018-12-18
Filing date: 2018-12-18
Publication date: 2020-07-08
Also published as: GB201820614D0

Abstract

A vaccine composition comprising a peptide comprising a CD8+ T-cell epitope as set out in SEQ ID No.s 1-43 as define herein. The CD8+ T cell epitope may be capable of interacting with two different HLA super-types. The vaccine composition may comprise two or more hepatitis B viral (HBV) peptides, each comprising a different CD8+ T cell epitope. The peptide or peptides may be attached to a nanoparticle. The vaccine composition may comprise a peptide comprising a CD4+ epitope. The CD4+ T cell epitope may be capable of interacting with all HLA class II subtypes. The vaccine composition may be used to treat a HBV infection of serotype adr, adw, ayr, or ayw. The vaccine composition may be used to treat a HBV infection of genotype A, B, C, D, E, F, G, H, I, or J.

Description

MHC CLASS I ASSOCIATED PEPTIDES FOR PREVENTION AND TREATMENT OF HEPATITIS B VIRUS INFECTION

Field of the invention

The invention relates to vaccine compositions comprising hepatitis B virus peptides, and the use of such compositions for the treatment and prevention of hepatitis B virus infection.

Background to the invention

Hepatitis B virus (HBV) is a member of the Hepadnaviridae family of viruses which also includes woodchuck hepatitis virus (WHY) and duck hepatitis B virus. These viruses are primarily hepatotropic with infections characterized by fever, fatigue, muscle aches, and yellowing of the eyes and/or skin. The severity of these symptoms can vary with a proportion of cases being asymptomatic. More than 2.5 billion people worldwide have been infected by HBV, but for the vast majority of adults encountering the virus (>90%), the infection is acute and readily cleared by the immune system. For the remaining 5-10% of adults, and for neonates and unvaccinated children, HBV establishes a chronic infection. Approximately 370 million people worldwide are chronically infected and over 500,000 people die each year due to complications from HBV. These complications include liver cirrhosis, liver failure, and/or hepatocellular carcinoma (HCC) and it is estimated that up to 40% of chronically infected patients will develop at least one of these complications.

The primary determinant of whether hepatitis B virus is cleared or establishes a chronic infection is the robustness of the immune response, in particular the CD8+ T cell response. Data from both animal models and infected patients indicate that strong innate immune responses are crucial in controlling initial HBV replication and for subsequently activating the adaptive T cell response. In patients that resolve acute infections, there are greater numbers of IFN-y secreting CD4+ and CD8+ T cells with a broader range of epitope recognition than in chronically infected patients. Although individuals that initially fail to mount vigorous T cell responses develop chronic infection, data indicate that virus specific T cells are still capable of a broad, effective T cell response. Rehermann et al. demonstrated that a small number of chronically infected individuals mount robust CTL responses against HBV either spontaneously or in response to IFN-a treatment. These T cells are directed against multiple proteins indicating that chronically infected patients can also mount a broad response to viral antigens. These data suggest that therapeutic interventions designed to stimulate robust and multi-epitope specific responses may be sufficient to resolve chronic HBV infections. Yet, despite an effective prophylactic vaccine, the development of therapies capable of eliminating HBV from chronically infected individuals has proven challenging. Thus, there is a critical need for targeted therapeutic vaccines capable of inducing robust, sustained T cell responses capable of permanent clearance of virus.

Summary of the invention

The present invention relates to a hepatitis B virus vaccine composition that stimulates an immune response while avoiding the adverse clinical effects often associated with vaccines containing viruses. The vaccine composition may provide protection against multiple serotypes of hepatitis B virus (e.g. serotype adr, serotype adw, serotype ayr and/or serotype ayw) and/or multiple genotypes or subgenotypes of hepatitis B virus (e.g. genotype A, genotype B, genotype C, genotype D, genotype E, genotype F, genotype G, genotype H and/or genotype J).

The present inventors have surprisingly identified that a nanoparticle, for example a gold nanoparticle, may be used to induce an efficient response to a vaccine composition designed to stimulate a T cell response against a hepatitis B virus. Use of a nanoparticle abrogates the need to use a virus in the vaccine composition. The use of a traditional adjuvant, which may be associated with adverse reactions in the clinic, is also avoided. Therefore, the likelihood of an individual experiencing an adverse reaction following administration of the vaccine composition is reduced.

The present inventors have also identified number of peptides that are conserved between different hepatitis B viruses and are presented by IVIHC molecules on cells infected with those viruses. Inclusion of such conserved peptides in the vaccine composition may confer protective capability against multiple serotypes, genotypes and/or subgenotypes of hepatitis B virus. Including multiple conserved peptides that bind to different HLA supertypes in the vaccine composition results in a vaccine that is effective in individuals having different HLA types.

Accordingly, the present invention provides a vaccine composition comprising a peptide comprising one or more of the CD8+ T cell epitopes set out in SEQ ID NOs: I to 43SEQ ID NOs: 1 to 43. In some aspects, the hepatitis B virus peptide may be attached to a nanoparticle.

The present invention further provides: -a method of preventing or treating a hepatitis B virus infection, comprising administering the vaccine composition of the invention to an individual infected with, or at risk of being infected with, a hepatitis B virus; and -a vaccine composition of the invention for use in a method of preventing or treating a hepatitis B virus infection in an individual.

Detailed Description of the Invention

Vaccine compositions The present invention provides a vaccine composition comprising a hepatitis B virus peptide comprising one or more of the CD8+ T cell epitopes set out in SEQ ID NOs: 1 to 43. This vaccine composition has a number of benefits which will become apparent from the discussion below. The key benefits are though summarised here.

Firstly, the vaccine composition of the invention advantageously comprises a hepatitis B virus peptide comprising one or more of the CD8+ T cell epitopes set out in SEQ ID NOs: 1 to 43 and newly identified by the inventors. The vaccine composition is therefore capable of stimulating a cellular immune response (e.g. a CD8+ T cell response) against a hepatitis B virus. CD8+ cytotoxic T lymphocytes (CTLs) mediate viral clearance via their cytotoxic activity against infected cells. Stimulating cellular immunity may therefore provide a beneficial defence against hepatitis B virus infection.

Secondly, inclusion in the vaccine composition of one or more of the CD8+ T cell epitopes set out in SEQ ID NOs: t to 43 may aid in the clearance of hepatitis B virus from chronically infected individuals. The present inventors used an immunoproteomic approach to identify MHC class I peptides presented by cells chronically infected with hepatitis B virus. This approach has distinct advantages over traditional vaccine design algorithms as it identifies antigens naturally processed and presented by infected cells. Accordingly, the vaccine composition of the invention allows a chronically infected individual to be directly administered with a CD8+ T cell epitope relevant to their infection, facilitating activation of a T cell response against the hepatitis B virus. In contrast, live, attenuated or recombinant viruses contained in traditional vaccine compositions must be biologically processed to form A/MC-associated epitopes before they are capable of stimulating an immune response. The epitopes generated by processing may not accurately reflect those epitopes relevant to chronic infection, meaning that a T cell response induced by a traditional, virus-based vaccine may not be correctly targeted to chronically infected cells.

Thirdly, a number of the CD8+ T cell epitopes identified by the present inventors may be conserved between different hepatitis B viruses, and may be presented by MHC molecules on cells infected with those viruses. Inclusion of such conserved peptides in the vaccine composition may confer protective capability against multiple serotypes, genotypes and/or subgenotypes of hepatitis B virus, i.e. confer cross-protection. 100% homology between hepatitis B viruses is not required for cross-protection to be conferred.

Rather, cross-protection may arise following immunisation with a sequence that is, for example, about 50% or more (such as 60%, 70%, 75%, 80%, 90%, 95%, 98% or 99%) homologous to a CD8+ T cell epitope expressed in a cell infected with hepatitis B virus, if certain residues are retained in the correct position. A vaccine composition comprising one or more CD8+ T cell epitopes set out in SEQ ID NOs: 1 to 43 may therefore be capable of providing cross-protection against a wide variety of existing hepatitis B viruses. Inclusion of one or more conserved peptides in the vaccine composition may also confer protective capability against emerging hepatitis B virus strains associated with evolution of the hepatitis B virus genome. In this way, a single hepatitis B virus vaccine composition can be used to confer protection against a variety of different hepatitis B viruses. This provides a cost-effective means of controlling the spread of hepatitis B virus infection.

Fourthly, different CD8+ T cell epitopes identified by the present inventors are capable of binding to different HLA supertypes. Inclusion of multiple peptides each comprising a CD8+ T cell epitope capable of binding to a different HLA supertype results in a vaccine composition that is effective in individuals having different HLA types. In this way, a single hepatitis B virus vaccine composition can be used to confer protection in a large proportion of the human population. This again provides a cost-effective means of controlling the spread of hepatitis B virus infection.

Fifthly, the hepatitis B virus peptide comprised in the vaccine composition of the invention may be attached to a nanoparticle, for example a gold nanoparticle. As described in more detail below, attachment to a nanoparticle reduces or eliminates the need to include an adjuvant in the vaccine composition. Thus, the vaccine composition of the invention is less likely to cause adverse clinical effects upon administration to an individual.

Peptides The vaccine composition of the invention comprises a hepatitis B virus peptide comprising one or more of the CD8+ T cell epitopes set out in SEQ ID NOs: 1 to 43. The vaccine composition may comprise from about one to about 50 such peptides, such as about 2 to 40, 3 to 30, 4 to 25, 5 to 20, 6 to 15, 7, 8, 9 or 10 such peptides. SEQ ID NOs: 1 to 43 are set out in Table 1.

Epitope HLA Parent Protein Accession ID SEQ ID binding NO: ADHGARLSL X protein [Hepatitis B virus] gi14211488 1 AICSVLRRA A2 polymerase [Hepatitis B virus] gi224978740 2 ALMPIYTCI A2/24 polymerase [Hepatitis B virus] gi452628 3 CFEHGEHHI A24 Polymerase [Hepatitis B virus] g 222824656 4 DLVPNIMENI A2/24 middle S protein [Hepatitis B virus] gi209362964 5 DNQYGTMQNLQN polymerase [Hepatitis B virus] gil 12380514 6 ELGEENRLMIYVL A2/24 X protein [Hepatitis B virus] gi83314087 7 FDIRASSASS polymerase [Hepatitis B virus] gi190147544 8 FGATVET,T.TF 1-1BcAb [Hepatitis B virus] gi306268 9 FLDPLLVLQA A2 HBsAg [Hepatitis B virus] gi58190162 10 GILTTVPTV A2 S protein [Hepatitis B virus] gi38198572 11 GRDLLDTARA precore/core ORF [Hepatitis B virus] 0971629 12 GRPFSGPPGAL X peptide [Hepatitis B virus] g 198447151 13 GRPLPGPLGAVS Hbx protein [Hepatitis B virus] gi108937192 14 GSPLSWEQELQ P protein [Hepatitis B virus] gi164416627 15 IFARIGDPA large S protein [Hepatitis B virus] gi209484443 16 ILLPMLIFLLV A2 middle S protein [Hepatitis B virus] g 2289202 17 ILTTMPAAPPSA A2 large surface protein [Hepatitis B virus] g 205362291 18 INNLANKSAS polymerase [Hepatitis B virus] gil 1935073 19 ISSISSRTGDL S protein [Hepatitis B virus] gi38198448 20 ISTIPQSLDS surface antigen [Hepatitis B virus] gi3328369 21 LLDLKGMLP S protein [Hepatitis B virus] gi157411138 22 LLTKILII A2/24 S protein [Hepatitis B virus] g 205986003 23 LNSLVPFVQ S protein [Hepatitis B virus] gi189171098 24 LYKWTLGL A24 X protein [Hepatitis B virus] gi27466468 25 LYNILSPFLL A24 S protein [Hepatitis B virus] gi157411152 26 METTVNAPLN X protein [Hepatitis B virus] gi193248379 27 MLASSNTSRGTC A3 S protein [Hepatitis B virus] gi 162424181 28 NFLGGTIECL A24 S protein [Hepatitis B virus] 81225632851 29 NINYHHET A2 polymerase [Hepatitis B virus] g 112949722 30 PPASPPIVPS X peptide [Hepatitis B virus] gi6692552 31 QSKGPVLSCWLL polymerase [Hepatitis B virus] gi55274830 32 S SIYKRIGDP large surface protein [Hepatitis B virus] g 205362323 33 SSKPRKGIVIVT preSlipreS2 surface [Hepatitis B virus] gi51449942 34 TKPLLGNCTCIPI S protein [Hepatitis B virus] gi167507457 35 TLPNLHDNCSRNL polymerase [Hepatitis B virus] gi146746963 36 TVRKAADPA A2 polymerase [Hepatitis B virus] gi198076006 37 VLLGFAAPFTQ A24 polymerase [Hepatitis B virus] gi164654403 38 VTNLQSLTNLLS A2 polymerase [Hepatitis B virus] g 39748644 39 WVGSNLTFGREIV A24 core protein [Hepatitis B virus] g 226713196 40 WYLGPSLYSIVSL A24 S protein [Hepatitis B virus] gi 164416610 41 YPGIQAIQA polymerase [Hepatitis B virus] gi224810371 42 YVDDVVLGAESV A2 reverse transcriptase [Hepatitis B virus] g 76251861 43 FLGGPPVCL A2/24 Surface (S) Q OEED2 44 ILRSFIPLL A2/24 Surface (S) Q6WYY8 45 FLKQQYMNL A2/24 Polymerase (P) IODE20 46 FLSKQYMDL A2/24 Polymerase (P) L7QBE1 47 TVSTKLCKI A2/24 Polymerase (P) Q8B4E6 48 GGPNLDNIL Large E Q8QSF2 49 LTTVPAASLLA A2/24 Large E Q9YKJ7 50 LTFGRETVLEN A2/24 Precore/Core (C) Q6UFV9 51 IYDHQHGTL A24 Polymerase (P) C9ED71 52 TVLENLVSLGV A2/24 precore/core protein [Hepatitis B virus] gi34419971 53 QANIQWNSLAF A2 large S protein [Hepatitis B virus] g 222824657 54 IEANKVGV preS1 protein [Hepatitis B virus] gi 164509420 55 AQGTLTSVPV large S protein [Hepatitis B virus] gi283971254 56 VLLDCQGMLL A2/24 hepatitis surface antigen [Hepatitis B virus] g 94466780 57 MAARLRCQL A2 X protein [Hepatitis B virus] gi260184403 58 KVCRRIVGLLGFA A2/24 polymerase [Hepatitis B virus] gi225675980 59 NTNMGLKILQLLW A2/24 pre-C/C protein [Hepatitis B virus] gi164416566 60

Table 1

The hepatitis B virus peptide comprising one or more of the CD8+ T cell epitopes set out in SEQ ID NOs: 1 to 43 may consist of a CD8+ T cell epitope set out in SEQ ID NOs: 1 to 43. The hepatitis B virus peptide may comprise only one of the CD8+ T cell epitopes set out in SEQ ID NOs: 1 to 43. The hepatitis B virus peptide may comprise two or more, such as three or more, four or more, five or more, six or more, seven or more, eight or more, nine or more, ten or more, 11 or more, 12 or more, 13 or more, 14 or more, 15 or more, 16 or more, 17 or more, 18 or more, 19 or more, 20 or more, 21 or more, 22 or more, 23 or more, 24 or more, 25 or more, 26 or more, 27 or more, 28 or more, 29 or more, 30 or more, 31 or more, 32 or more, 33 or more, 34 or more, 35 or more, 36 or more, 37 or more, 38 or more, 39 or more, 40 or more, 41 or more, or 42 or more of the CD8+ T cell epitopes set out in SEQ ID NOs: 1 to 43, in any combination. The hepatitis B virus peptide comprising one or more of the CD8+ T cell epitopes set out in SEQ ID NOs: 1 to 43 may comprise all of the CD8+ T cell epitopes set out in SEQ ID NOs: 1 to 43.

As well as one or more of the CD8+ T cell epitopes set out in SEQ ID NOs: 1 to 43, the hepatitis B virus peptide may comprise one or more other CD8+ T cell epitopes, one or more CD4+ T cell epitopes and/or one or more B cell epitopes. For example, the hepatitis B virus peptide may comprise two or more, such as three or more, four or more, five or more, ten or more, fifteen or more, or twenty or more CD8+ T cell epitopes other than those set out in SEQ ID NOs: 1 to 43. For example, the hepatitis B virus peptide may comprise one or more (such as two or more, three or more, four or more, five or more, six or more, seven or more, eight or more, nine or more, ten or more, 11 or more, 12 or more, 13 or more, 14 or more, 15 or more, 16 or more, or 17) of the CD8+ T cell epitopes set out in SEQ ID NOs: 44 to 60, in any combination. The hepatitis B virus peptide may comprise all of the CD8+ T cell epitopes set out in SEQ ID NOs: 44 to 60. The hepatitis B virus peptide may comprise two or more, such as three or more, four or more, five or more, ten or more, fifteen or more, or twenty or more CD4+ T cell epitopes. The hepatitis B virus peptide may comprise two or more, such as three or more, four or more, five or more, ten or more, fifteen or more, or twenty or more B cell epitopes.

The vaccine composition may comprise two or more hepatitis B peptides each comprising a different CD8+ T cell epitope. For instance, the vaccine composition may comprise two or more hepatitis B virus peptides each comprising a CD8+ T cell epitope comprising a different sequence selected from SEQ ID NOs: 1 to 43. The vaccine composition may comprise two or more hepatitis B virus peptides each comprising a CD8+ T cell epitope comprising a different sequence selected from SEQ ID NOs: 1 to 60. Each of the hepatitis B virus peptides may have any of the properties set out in the preceding paragraphs. For instance, each hepatitis B virus peptide may comprise multiple CD8+ T cell epitopes set out in SEQ ID NOs: 1 to 43 and, optionally, one or more other CD8+ T cell epitopes, one or more CD4+ T cell epitopes and/or one or more B cell epitopes. In one aspect, the vaccine composition may comprise three or more, four or more, five or more, six or more, seven or more, eight or more, or nine or more hepatitis B virus peptides each comprising a CD8+ T cell epitope comprising a different sequence selected from SEQ ID NOs: 1 to 43. The vaccine composition may, for example, comprise 43 hepatitis B virus peptides each comprising a CD8+ T cell epitope comprising a different sequence selected from SEQ ID NOs: 1 to 43. In one aspect, the vaccine composition may comprise three or more, four or more, five or more, six or more, seven or more, eight or more, or nine or more hepatitis B virus peptides each comprising a CD8+ T cell epitope comprising a different sequence selected from SEQ ID NOs: 1 to 60. The vaccine composition may, for example, comprise 60 hepatitis B virus peptides each comprising a CD8+ T cell epitope comprising a different sequence selected from SEQ ID NOs: 1 to 60.

The vaccine composition may further comprise one or more (such as about 1 to 50, 2 to 40, 3 to 30, 4 to 25, 5 to 20, 6 to 15, 7, 8, 10 or 10) additional peptides each comprising one or more epitopes. The epitope may be a CD8+ T cell epitope, a CD4+ T cell epitope and/or a B cell epitope. The CD8+ T cell epitope is preferably a CD8+ T cell epitope other than the CD8+ T cell epitopes set out in SEQ ID NOs: 1 to 60. The CD8+ T cell epitope may, for example, be a hepatitis B virus CD8+ epitope, i.e. a peptide that is expressed by one or more hepatitis B viruses and that is that is capable of (i) presentation by a class I MEIC molecule and (ii) recognition by a T cell receptor (TCR) present on a CD8+ T cell. Alternatively, the CD8+ T cell epitope may be an CD8+ T cell epitope that is not expressed by one or more hepatitis B viruses. The CD4+ T cell epitope may, for example, be a hepatitis B virus CD4+ epitope, i.e. a peptide that is expressed by one or more hepatitis B viruses and that is that is capable of (i) presentation by a class II MHC molecule and (ii) recognition by a T cell receptor (TCR) present on a CD4+ T cell. Alternatively, the CD4+ T cell epitope may be a CD4+ T cell epitope that is not expressed by one or more hepatitis B viruses. CD8+ and CD4+ T cell epitopes are described in more detail below.

A hepatitis B virus peptide is a peptide that is expressed by one or more hepatitis B viruses. Numerous species of hepatitis B virus exist. There are four major serotypes of hepatitis B virus (adr, adw, ayr, and ayw) based on antigenic epitopes present on the virus's envelope proteins. These serotypes are based on a common determinant (a) and two mutually exclusive determinant pairs (d/y and w/r). Strains of hepatitis B virus have also been divided into ten genotypes (genotype A, genotype B, genotype C, genotype D, genotype E, genotype F, genotype G, genotype H and genotype J) and forty subgenotypes according to nucleotide sequence variation of the genome.

Any hepatitis B virus peptide comprised in the vaccine composition of the invention may comprise a peptide that is expressed by one or more of serotype adr, serotype adw, serotype ayr, serotype ayw, genotype A, genotype B, genotype C, genotype D, genotype E, genotype F, genotype G, genotype H and genotype J hepatitis B virus. For example, a hepatitis B virus peptide comprised in the vaccine composition of the invention may comprise a peptide that is expressed by serotype adr, serotype adw, serotype ayr, or serotype ayw hepatitis B virus. A hepatitis B virus peptide comprised in the vaccine composition of the invention may comprise a peptide that is expressed by genotype A, genotype B, genotype C, genotype D, genotype E, genotype F, genotype G, genotype H and genotype J hepatitis B virus. When the composition comprises an additional peptide that is a hepatitis B virus peptide, that additional hepatitis B virus peptide may be expressed by one or more of serotype adr, serotype adw, serotype ayr, serotype ayw, genotype A, genotype B, genotype C, genotype D, genotype E, genotype F, genotype G, genotype I-I and genotype J hepatitis B virus. Accordingly, the vaccine composition may comprise hepatitis B virus peptides from one or more hepatitis B viruses, such as 1 to 20, 2 to 19, 3 to 18, 4 to 17, 5 to 16, 6 to 15, 7 to 14, 8 to 13, 9 to 12, 10 or 11 hepatitis B viruses.

The peptide may be expressed one or more of (1) serotype adr, (2) serotype adw, (3) serotype ayr, or (4) serotype ayw hepatitis B virus in any combination such as, for example: 1; 2; 3; 4; 1 and 2; 1 and 3; 1 and 4; 2 and 3; 2 and 4; 3 and 4; 1, 2 and 3; 1, 2 and 4; 1, 3 and 4; 2, 3 and 4; or 1, 2, 3 and 4. When a hepatitis B virus peptide comprised in the vaccine composition of the invention comprises a peptide that is expressed by genotype A, genotype B, genotype C, genotype D, genotype E, genotype F, genotype G, genotype H or genotype J, the peptide may be expressed by one or more these viruses alone or in any combination.

The hepatitis B virus peptide may be a peptide that is expressed on the surface of one or more hepatitis B viruses, or intracellularly within one or more hepatitis B viruses. The peptide may be a structural peptide or a functional peptide, such as a peptide involved in the metabolism or replication of the hepatitis B virus. Preferably, the peptide is an internal peptide. Preferably, the peptide is conserved between two or more different hepatitis B viruses, hepatitis B virus serotypes or hepatitis B virus genotypes. A peptide is conserved between two or more different hepatitis B viruses, hepatitis B virus serotypes or hepatitis B virus genotypes if each of the two or more different hepatitis B viruses, hepatitis B virus serotypes or hepatitis B virus genotypes encodes a sequence that is 50% or more (such as 60%, 70%, 75%, 80%, 90%, 95%, 98% or 99%) homologous to the peptide.

The hepatitis B virus peptide may contain any number of amino acids, i.e. be of any length. Typically, the hepatitis B virus peptide is about 8 to about 30, 35 or 40 amino acids in length, such as about 9 to about 29, about 10 to about 28, about 11 to about 27, about 12 to about 26, about 13 to about 25, about 13 to about 24, about 14 to about 23, about 15 to about 22, about 16 to about 21, about 17 to about 20, or about 18 to about 29 amino acids in length.

The hepatitis B virus peptide may be chemically derived from a polypeptide hepatitis B virus antigen, for example by proteolytic cleavage. More typically, the hepatitis B virus peptide may be synthesised using methods well known in the art.

The term "peptide" includes not only molecules in which amino acid residues are joined by peptide (-CO-NH-) linkages but also molecules in which the peptide bond is reversed. Such retro-inverso peptidomimetics may be made using methods known in the art, for example such as those described in Meziere et al (1997) J. Immuno1.159, 3230- 3237. This approach involves making pseudopeptides containing changes involving the backbone, and not the orientation of side chains. Meziere et al (1997) show that, at least for MI-IC class 11 and T helper cell responses, these pseudopeptides are useful. Retroinverse peptides, which contain NH-CO bonds instead of CO-NH peptide bonds, are much more resistant to proteolysis.

Similarly, the peptide bond may be dispensed with altogether provided that an appropriate linker moiety which retains the spacing between the carbon atoms of the amino acid residues is used; it is particularly preferred if the linker moiety has substantially the same charge distribution and substantially the same planarity as a peptide bond. It will also be appreciated that the peptide may conveniently be blocked at its N-or C-terminus so as to help reduce susceptibility to exoproteolytic digestion. For example, the N-terminal amino group of the peptides may be protected by reacting with a carboxylic acid and the C-terminal carboxyl group of the peptide may be protected by reacting with an amine. Other examples of modifications include glycosylation and phosphorylation. Another potential modification is that hydrogens on the side chain amines of R or K may be replaced with methylene groups (-NH2 may be modified to -NH(Me) or -N(Me)z).

The term "peptide" also includes peptide variants that increase or decrease the half-life of the peptide in vivo. Examples of analogues capable of increasing the half-life of peptides used according to the invention include peptoid analogues of the peptides, D-amino acid derivatives of the peptides, and peptide-peptoid hybrids. A further embodiment of the variant polypeptides used according to the invention comprises D-amino acid forms of the polypeptide. The preparation of polypeptides using D-amino acids rather than L-amino acids greatly decreases any unwanted breakdown of such an agent by normal metabolic processes, decreasing the amounts of agent which needs to be administered, along with the frequency of its administration.

C1)8+ T cell epitopes The vaccine composition of the invention comprises a hepatitis B virus peptide comprising one or more of the CD8+ T cell epitopes set out in SEQ ID NOs: 1 to 43 (see Table 1) or a variant thereof. The hepatitis B virus peptide may further comprise one or more (such as two or more, three or more, four or more, five or more, ten or more, fifteen or more, or twenty or more) other CD8+ T cell epitopes. The vaccine composition may further comprise one or more (such as 1 to 50, 2 to 40, 3 to 30, 4 to 25, 5 to 20, 6 to 15, 7, 8, 9 or 10) additional peptides each comprising one or more CD8+ T cell epitopes. Preferably, the additional peptide is a hepatitis B virus peptide.

A CD8+ T cell epitope is a peptide that is capable of (i) presentation by a class I MHC molecule and (ii) recognition by a T cell receptor (TCR) present on a CD8+ T cell.

Preferably, recognition by the TCR results in activation of the CD8+ T cell. CD8+ T cell activation may lead to increased proliferation, cytokine production and/or cyotoxic effects. Typically, the CD8+ T cell epitope is around 9 amino acids in length. The CD8+ T cell epitope may though be shorter or longer. For example, the CD8+ T cell epitope may be about 8, 9, 10, 11, 12, 13, 14 or 15 amino acids in length. The CD8+ T cell epitope may be about 8 to 15, 9 to 14 or 10 to 12 amino acids in length.

Hepatitis B virus peptides comprising a CD8+ T cell epitope are known in the art. Methods for identifying CD8+ T cell epitopes are known in the art. Epitope mapping methods include X-ray co-crystallography, array-based oligo-peptide scanning (sometimes called overlapping peptide scan or pepscan analysis), site-directed mutagenesis, high throughput mutagenesis mapping, hydrogen-deuterium exchange, crosslinking coupled mass spectrometry, phage display and limited proteolysis. MHC motif prediction methodologies may also be used.

CD8+ T cell epitopes presented by hepatitis B virus-infected cells can be identified in order to directly identify CD8+ T cell epitopes for inclusion in the vaccine composition.

This is an efficient and logical method which can be used alone or to confirm the utility of potential CD8+ T cell epitopes identified by MHC motif prediction methodologies. This method was used by the inventors to identify the CD8+ T cell epitopes set out in SEQ ID NOs: 1 to 43 (see Example 1).

To perform the method, cells are infected with a hepatitis B virus and maintained in culture for a period of around 72 hours at a temperature of around 37°C. Following culture, the cells are then harvested and washed. Next, the cells are lysed, for instance by homogenisation and freezing/thawing in buffer containing 1% NP40. Lysates are cleared by centrifugation at 2000rpm for 30 minutes to remove cell debris.

MHC/peptide complexes are then isolated from the lysates by immunoaffinity chromatography using protein A/G beads (UltraLink Immobilized Protein A/G, pierce, Rockford, IL) coated with W6/32 (a monoclonal antibody recognising pan MHC class I molecule). To coat the beads with the antibody, the beads are washed with low pH buffer followed by PBS rinses, incubated with 0.5mg of the antibody at room temperature for 2 hours, and washed three times to remove unbound antibody. For immunoaffinity chromatography, the coated beads are incubate with lysate for 2 hours at room temperature with continuous rocking. The beads are then separated from the lysate by centrifuging at 1000 rpm for 5 minutes. Bound MHC complexes are eluted from the beads by the addition of 0.1% trifluoroacetic acid (TFA), pH 1.5.

The eluate is next heated at 85°C for 15 minutes to dissociate the bound peptides from the MHC molecules. After cooling to room temperature, peptides are separated from the antibody by centrifugation using, for example, 3 kDa molecular mass cutoff membrane filters (Millipore). The filtrate is concentrated using vacuum centrifugation and reconstituted to a final volume of 1000. The purified peptide mixture is fractionated, for example using a C-18 reversed phase (RP) column (e.g. 4.6 mm diameter x 150 mm length) using an offline HPLC. For this step, mobile phase A may be 2% acetonitrile (CAN) and 0.1% formic acid (FA) in water, while mobile phase B may be 0.1% FA and 90% CAN in water.

The peptide-containing fractions are then eluted from the column, dried under a vacuum, and analysed by mass spectrometry to identify the sequences of the fractions. The acquired spectral data can then be searched against all databased hepatitis B virus proteins to identify peptide sequences associated with hepatitis B virus. Synthetic peptides may then be made according to the identified sequences and subjected to mass spectrometry to confirm their identity to the peptides in the peptide-containing fractions.

In this method, any type of cells may be infected with hepatitis B virus. The cells may be antigen presenting cells. The cells may be hepatoma cells such as HepG2 cells, EBV-transformed lymphoblasto d B cells such as TY cells, or lymphoblasts such as T2 cells.

Likewise, any hepatitis B virus of interest may be used to infect the cells. For instance, the hepatitis B virus may be a serotype adr, serotype adw, serotype ayr, serotype ayw, genotype A, genotype B, genotype C, genotype D, genotype E, genotype F, genotype G, genotype H or genotype J hepatitis B virus.

The direct identification of CD8+ T cell epitopes presented by hepatitis B virus-infected cells is advantageous compared to IMHC motif prediction methodologies. The immune epitope database (IEDB; http://ww-wledb.org) is generated by motif prediction methods, and not functional methods, and contains numerous predicted HLA-specific hepatitis B virus T cell epitopes, including some shared epitopes with high MHC binding scores and limited CTL characterization. As both dominant and subdominant epitopes may be presented by hepatitis B virus-infected cells, it is difficult to sort out the dominance hierarchies of naturally presented epitopes using the database. Thus, it is not clear from the immune epitope database alone which of the listed epitopes may be expected to efficiently induce a CD8+ T cell response when included in a vaccine composition. The direct identification method set out above provides a mechanism for confirming the utility of the epitopes.

Vaccine compositions based on epitopes presented by hepatitis B virus-infected cells, such as the vaccine composition of the invention, are superior to vaccines based on a viral protein subunit or a motif predicted epitope. Protein processing by the immune system is likely to alter native viral epitopes. Basing a vaccine composition on peptides demonstrated to be presented by infected cells removes this source of uncertainty, because the peptides have already undergone protein processing.

Furthermore, the direct identification method may be used to identify conserved CD8+ T cell epitopes presented by cells infected by different hepatitis B viruses. In this way, CD8+ T cell epitopes suitable for inclusion in a cross-protective vaccine may be identified.

Cross-protective vaccine compositions The vaccine composition of the invention is designed to elicit a protective immune response against hepatitis B virus infection. The vaccine composition may also induce cross-protection against a wide range of hepatitis B viruses, as the SEQ ID NOs: 1 to 43 are highly conserved between hepatitis B viruses.

An immune response generated by vaccination with a composition that comprises an epitope that is 100% homologous with a sequence from another hepatitis B virus may protect against subsequent infection with that hepatitis B virus. An immune response generated by vaccination with a composition that comprises an epitope that is about 50% or more (such as 60%, 70%, 75%, 80%, 90%, 95%, 98% or 99%) homologous with a sequence encoded by another hepatitis B virus may protect against subsequent infection with that hepatitis B virus. In some cases, the protective effect is associated with the conservation of certain residues between the epitope and the sequence encoded by the other hepatitis B virus. Immunisation with a vaccine composition of the invention may therefore induce a protective immune response against a wide variety of hepatitis B viruses.

Accordingly, the vaccine composition of the invention may have built-in crossserotype and/or cross-genotype efficacy, i.e. be a cross-protective hepatitis B virus vaccine composition. In this way, a single hepatitis B virus vaccine composition can be used to confer protection against a variety of different hepatitis B viruses. This provides a cost-effective means of controlling the spread of hepatitis B virus infection.

Inclusion of conserved peptides in the vaccine composition may confer protective capability against emerging hepatitis B virus strains associated with evolution of the hepatitis B virus genome. This may assist in the long-term control of the hepatitis B virus 20 infection.

Interaction with HLA supertipes The vaccine composition may comprise at least two hepatitis B virus peptides comprising a CD8+ T cell epitope which each interacts with a different HLA supertype.

Including a plurality of such peptides in the vaccine composition allows the vaccine composition to elicit a CD8+ T cell response in a greater proportion of individuals to which the vaccine composition is administered. This is because the vaccine composition should be capable of eliciting a CD8+ T cell response in all individuals of an HLA supertype that interacts with one of the CD8+ T cell epitopes comprised in the plurality of hepatitis B virus peptides. Each CD8+ T cell epitope may interact with HLA-Al, HLAA2, HLA-A3, HLA-A24, HLA-B7, HLA-B8, HLA-B27, HLA-B44, HLA-B58 or HLAB62, or any other HLA supertype know in the art. Any combination of hepatitis B virus peptides comprising such a CD8+ T cell epitope is possible.

The vaccine composition may comprise at least one hepatitis B virus peptide comprising a CD8+ T cell epitope which interacts at least two different HLA supertypes. Again, this allows the vaccine composition to elicit a CD8+ T cell response in a greater proportion of individuals to which the vaccine composition is administered. The vaccine composition may comprise at least two, at least three, at least four, at least five, at least two, at least fifteen, or at least twenty hepatitis B virus peptides comprising a CD8+ T cell epitope that each interact with at least two different HLA subtypes. Each hepatitis B virus peptide may interact with at least two, at least three, at least four, at least five, at least six, at least 7, at least 8, at least 9 or at least 10 different HLA supertypes. Each hepatitis B virus peptide may interact with two or more of HLA-A1, HLA-A2, HLA-A3, HLA-A24, HLA-B7, HLA-B8, HLA-B27, HLA-B44, HLA-B58 or HLA-B62, or any other HLA supertype known in the art, in any combination. Preferably, the vaccine composition comprises a hepatitis B virus peptide comprising a CD8+ T cell epitope that interacts with HLA-A2 and HLA-24. In this case, the vaccine composition may, for example, comprise a hepatitis B virus peptide comprising a CD8+ T cell set out in SEQ ID NO: 3, 5, 7, or 23.

The vaccine composition may, for example, comprise a hepatitis B virus peptide comprising a CD8+ T cell set out in SEQ ID NO: 44, 45, 46, 47, 48, 50, 51, 53, 57, 59 or 60.

CD4 T cell epilopes The vaccine composition of the invention may comprise a peptide comprising a CD4+ T cell epitope. The vaccine composition may comprise two or more, such as three or more, four or more, five our more, ten or more, fifteen or more or twenty or more peptides comprising a CD4+ T cell epitope. A CD4+ T cell epitope is a peptide that is capable of (i) presentation by a class II MHC molecule and (ii) recognition by a T cell receptor (TCR) present on a CD4+ T cell. Preferably, recognition by the TCR results in activation of the CD4+ T cell. CD4+ T cell activation may lead to increased proliferation and/or cytokine production.

The CD4+ T cell epitope may be a hepatitis B virus CD4+ T cell epitope. That is, the CD4+ T cell epitope may be a peptide that is expressed by one or more hepatitis B viruses and that is that is capable of (i) presentation by a class 11 MHC molecule and (ii) recognition by a T cell receptor (TCR) present on a CD4+ T cell. Such peptides are known in the art.

The CD4+ T cell epitope may be a CD4+ T cell epitope other than a hepatitis B virus CD4+ T cell epitope. For example, the CD4+ T cell may be expressed by an organism other than a hepatitis B virus. The CD4+ T cell epitope may, for example, be expressed by Clostriudirtni tettmi. For instance, the CD4+ T cell epitope may be derived from tetanus toxin.

The CD4+ T cell epitope may be a CD4+ T cell epitope that reacts with all class II HLA types, i.e. a so-called "promiscuous" epitope. Inclusion of a promiscuous epitope in the vaccine composition may improve the ability of the vaccine composition to induce an immune response to the hepatitis B virus peptide comprising one or more of the CD8+ T cell epitopes set out in SEQ ID NOs: 1 to 43 SEQ ID NOs: 1 to 43. The CD4+ T cell epitope may, for example, comprise the sequence FKLQTMVKLFNRIKNNVA (SEQ ID NO: 61) and/or the sequence LQTMVKLFNRIKNNVAGGC (SEQ ID NO: 62). SEQ ID NOs 61 and 62 are promiscuous epitopes derived from tetanus toxin.

The peptide comprising a CD4+ T cell epitope may be a different peptide from the hepatitis B virus peptide comprising one or more of the CD8+ T cell epitopes set out in SEQ ID NOs: 1 to 43. The CD4+ T cell epitope may, for instance, be comprised in an additional peptide in the vaccine composition, i.e. in a peptide that does not comprise one or more of the CD8+ T cell epitopes set out in SEQ ID NOs: 1 to 43. As mentioned above, the additional peptide may comprise one or more CD8+ T cell epitopes and/or one or more B cell epitopes as well as the CD4+ T cell epitope. For instance, the additional peptide may comprise one or more hepatitis B virus CD8+ T cell epitopes.

The peptide comprising a CD4+ T cell epitope may be the same peptide as the hepatitis B virus peptide comprising one or more of the CD8+ T cell epitopes set out in SEQ ID NOs: 1 to 43. That is, the hepatitis B virus peptide comprising one or more of the CD8+ T cell epitopes set out in SEQ ID NOs: 1 to 43 may further comprise a CD4+ T cell epitope.

When the peptide comprising a CD4+ T cell epitope also comprises a CD8+ T cell epitope (such as one or more of the CD8+ T cell epitopes set out in SEQ ID NOs: 1 to 43 or one or more of the CD8+ T cell epitopes set out in SEQ ID NOs: 44 to 60), the CD8+ epitope may be nested within the CD4+ T cell epitope. CD4+ T cell epitopes are typically longer than CD8+ T cell epitopes. Therefore, extending one or both termini of the CD8+ T cell epitope may yield a longer, CD4+ T cell epitope whose sequence still comprises the CD8+ T cell epitope. Therefore, the CD4+ T cell epitope may comprise a CD8+ T cell epitope, such as a CD8+ T cell epitope set out in SEQ ID NOs: 1 to 43 or SEQ ID NOs: 44 to 60, extended at its N-terminus or C-terminus. The CD8+ T cell epitope may be extended by 1, 2, 3, 4 or 5 amino acids at its N terminus. The CD8+ T cell epitope may be extended by 1, 2, 3, 4 or 5 amino acids at its C terminus. Preferably, the CD8+ T cell epitope is extended by 3 amino acids at the N terminus, and 3 amino acids at the C terminus. However, the CD8+ T cell epitope need not be extended by the same number of amino acids at each terminus.

The CD8+ T cell epitope nested within a CD4+ T cell epitope may be capable of generating a robust CTL response. The extended peptide (CD4+ T cell epitope) may be capable of inducing T helper mediated cytokine responses. Thus, inclusion of a hepatitis B virus peptide comprising a CD8+ T cell epitope and a CD4+ T cell epitope in the vaccine composition may allow the vaccine composition to induce both cytotoxic and helper T cell responses.

The hepatitis B virus peptide comprising a CD4+ T cell epitope may be attached to a nanoparticle. When the peptide comprising a CD4+ T cell epitope is a different peptide from the hepatitis B virus peptide comprising one or more of the CD8+ T cell epitopes set out in SEQ ID NOs: 1 to 43or, each peptide may be attached to the same nanoparticle or to a different nanoparticle. The nanoparticle may be a gold nanoparticle. Nanoparticles and attachment thereto are described below.

B cell epitopes The vaccine composition of the invention may comprise a peptide comprising a B cell epitope. The vaccine composition may comprise two or more, such as three or more, four or more, five our more, ten or more, fifteen or more or twenty or more peptides comprising a B cell epitope. A B cell epitope is a peptide that is capable of recognition by a B cell receptor (BCR) present on a B cell. Preferably, recognition by the BCR results in activation and/or maturation of the B cell. B cell activation may lead to increased proliferation, and/or antibody production.

The B cell epitope may be a hepatitis B virus B cell epitope. That is, the B cell epitope may be a peptide that is expressed by one or more hepatitis B viruses and that is capable of recognition by a B cell receptor (BCR) present on a B cell. Such peptides are known in the art.

The B cell epitope may be a linear epitope, i.e. an epitope that is defined by the primary amino acid sequence of a particular region of a hepatitis B virus protein. Alternatively, the epitope may be a conformational epitope, i.e an epitope that is defined by the conformational structure of a native hepatitis B virus protein. In this case, the epitope may be continuous (i.e. the components that interact with the antibody are situated next to each other sequentially on the protein) or discontinuous (i.e. the components that interact with the antibody are situated on disparate parts of the protein, which are brought close to each other in the folded native protein structure).

Typically, the B cell epitope is around 5 to 20 amino acids in length, such as 6 to 19, 7 to 18, 8 to 17, 9 to 16, 10 to 15, 11 to 14 or 12 to 13 amino acids in length.

Methods for identifying B cell epitopes are also known in the art. For instance, epitope mapping methods may be used to identify B cell epitopes. These methods include structural approaches, wherein the known or modelled structure of a protein is be used in an algorithm based approach to predict surface epitopes, and functional approaches, wherein the binding of whole proteins, protein fragments or peptides to an antibody can be quantitated e.g. using an Enzyme-Linked Immunosorbent Assay (ELISA). Competition mapping, antigen modification or protein fragmentation methods may also be used.

Nanoparticles In the vaccine composition of the invention, the hepatitis B virus peptide comprising one or more of the CD8+ T cell epitopes set out in SEQ TD NOs: 1 to 43 may be attached to a nanoparticle. Any other peptides further comprised in the vaccine composition may also be attached to a nanoparticle. Attachment to a nanoparticle, for example a gold nanoparticle, is beneficial.

As set out above, attachment of the peptide to a nanoparticle (such as a gold nanoparticle) reduces or eliminates the need to include a virus or an adjuvant in the vaccine composition. The nanoparticles may contain immune "danger signals" that help to effectively induce an immune response to the peptides. The nanoparticles may induce dendritic cell (DC) activation and maturation, required for a robust immune response. The nanoparticles may contain non-self components that improve uptake of the nanoparticles and thus the peptides by cells, such as antigen presenting cells. Attachment of a peptide to a nanoparticle may therefore enhance the ability of antigen presenting cells to stimulate virus-specific T and/or B cells. Attachment to a nanoparticle also facilitates delivery of the vaccine compositions via the subcutaneous, intradermal, transdermal and oral/buccal routes, providing flexibility in administration.

Nanoparticles are particles between 1 and 100 nanometers (nm) in size which can be used as a substrate for immobilising ligands. In the vaccine compositions of the invention, the nanoparticle may have a mean diameter of 1 to 100, 20 to 90, 30 to 80, 40 to 70 or 50 to 60 nm. Preferably, the nanoparticle has a mean diameter of 20 to 40nm. A mean diameter of 20 to 40nm facilitates uptake of the nanoparticle to the cytosol. The mean diameter can be measured using techniques well known in the art such as transmission electron microscopy.

Nanoparticles suitable for the delivery of antigen, such as a hepatitis B virus peptide, are known in the art. Methods for the production of such nanoparticles are also known.

The nanoparticle may, for example, be a polymeric nanoparticle, an inorganic nanoparticle, a liposome, an immune stimulating complex (ISCOM), a virus-like particle (VLP), or a self-assembling protein. The nanoparticle is preferably a calcium phosphate nanoparticle, a silicon nanoparticle or a gold nanoparticle.

The nanoparticle may be a polymeric nanoparticle. The polymeric nanoparticle may comprise one or more synthetic polymers, such as poly(d,l-lactide-co-glycolide) (PLG), poly(d,l-lactic-coglycolic acid) (PLGA), poly(g-glutamic acid) (g-PGA)m poly(ethylene glycol) (PEG), or polystyrene. The polymeric nanoparticle may comprise one or more natural polymers such as a polysaccharide, for example pullulan, alginate, inulin, and chitosan. The use of a polymeric nanoparticle may be advantageous due to the properties of the polymers that may be include in the nanoparticle. For instance, the natural and synthetic polymers recited above may have good biocompatibility and biodegradability, a non-toxic nature and/or the ability to be manipulated into desired shapes and sizes. The polymeric nanoparticle may form a hydrogel nanoparticle. Hydrogel nanoparticles are a type of nano-sized hydrophilic three-dimensional polymer network. Hydrogel nanoparticles have favourable properties including flexible mesh size, large surface area for multivalent conjugation, high water content, and high loading capacity for antigens. Polymers such as Poly(L-lactic acid) (PLA), PLGA, PEG, and polysaccharides are particularly suitable for forming hydrogel nanoparticles. The nanoparticle may be an inorganic nanoparticle. Typically, inorganic nanoparticles have a rigid structure and are non-biodegradable. However, the inorganic nanoparticle may be biodegradable. The inorganic nanoparticle may comprise a shell in which an antigen may be encapsulated. The inorganic nanoparticle may comprise a core to which an antigen may be covalently attached. The core may comprise a metal. For example, the core may comprise gold (Au), silver (Ag) or copper (Cu) atoms. The core may be formed of more than one type of atom. For instance, the core may comprise an alloy, such as an alloy of Au/Ag, Au/Cu, Au/Ag/Cu, Au/Pt, Au/Pd or Au/Ag/Cu/Pd. The core may comprise calcium phosphate (CaP). The core may comprise a semiconductor material, for example cadmium selenide.

Other exemplary inorganic nanoparticles include carbon nanoparticles and silica-based nanoparticles. Carbon nanoparticles are have good biocompatibility and can be synthesized into nanotubes and mesoporous spheres. Silica-based nanoparticles (SiNPs) are biocompatible and can be prepared with tunable structural parameters to suit their therapeutic application.

The nanoparticle may be a silicon nanoparticle, such as an elemental silicon nanoparticle. The nanoparticle may be mesoporous or have a honeycomb pore structure. Preferably, the nanoparticle is an elemental silicon particle having a honeycomb pore structure. Such nanoparticles are known in the art and offer tunable and controlled drug loading, targeting and release that can be tailored to almost any load, route of administration, target or release profile. For example, such nanoparticles may increase the bioavailability of their load, and/or improve the intestinal permeability and absorption of orally administered actives. The nanoparticles may have an exceptionally high loading capacity due to their porous structure and large surface area. The nanoparticles may release their load over days, weeks or months, depending on their physical properties. Since silicon is a naturally occurring element of the human body, the nanoparticles may elicit no response from the immune system. This is advantageous to the in vivo safety of the nanoparticles.

Any of the SiNPs described above may be biodegradable or non-biodegradable. A biodegradable SiNP may dissolve to orthosilic acid, the bioavailable form of silicon. Orthosilic acid has been shown to be beneficial for the health of bones, connective tissue, hair, and skin.

The nanoparticle may be a liposome. Liposomes are typically formed from biodegradable, non-toxic phospholipids and comprise a self-assembling phospholipid bilayer shell with an aqueous core. A liposome may be an unilameller vesicle comprising a single phospholipid bilayer, or a multilameller vesicle that comprises several concentric phospholipid shells separated by layers of water. As a consequence, liposomes can be tailored to incorporate either hydrophilic molecules into the aqueous core or hydrophobic molecules within the phospholipid bilayers. Liposomes may encapsulate antigen within the core for delivery. Liposomes may incorporate viral envelope glycoproteins to the shell to form virosomes. A number of liposome-based products are established in the art and are approved for human use.

The nanoparticle may be an immune-stimulating complex (ISCOM). ISCOMs are cage-like particles which are typically formed from colloidal saponin-containing micelles.

1SCOMs may comprise cholesterol, phospholipid (such as phosphatidylethanolamine or phosphatidylcholine) and saponin (such as Quil A from the tree Quillaia saponaria). ISCOMs have traditionally been used to entrap viral envelope proteins, such as envelope proteins from herpes simplex virus type 1, hepatitis B, or influenza virus.

The nanoparticle may be a virus-like particle (VLP). VLPs are self-assembling nanoparticles that lack infectious nucleic acid, which are formed by self-assembly of biocompatible capsid protein. VLPs are typically about 20 to about 150nm, such as about 20 to about 40nm, about 30 to about 140nm, about 40 to about 130nm, about 50 to about 120nm, about 60 to about 110nm, about 70 to about 100nm, or about 80 to about 90nm in diameter. VLPs advantageously harness the power of evolved viral structure, which is naturally optimized for interaction with the immune system. The naturally-optimized nanoparticle size and repetitive structural order means that VLPs induce potent immune responses, even in the absence of adjuvant.

The nanoparticle may be a self-assembling protein. For instance, the nanoparticle may comprise ferritin. Ferritin is a protein that can self-assemble into nearly-spherical 10 nm structures. The nanoparticle may comprise major vault protein (MVP). Ninety-six units of MVP can self-assemble into a barrel-shaped vault nanoparticle, with a size of approximately 40 nm wide and 70 nm long.

The nanoparticle may be a calcium phosphate (CaP) nanoparticle. CaP nanoparticles may comprise a core comprising one or more (such as two or more, 10 or more, 20 or more, 50 or more, 100 or more, 200 or more, or 500 or more) molecules of CaP. CaP nanoparticles and methods for their production are known in the art. For instance, a stable nano-suspension of CAP nanoparticles may be generated by mixing inorganic salt solutions of calcium and phosphates in pre-determined ratios under constant mixing.

The CaP nanoparticle may have an average particle size of about 80 to about 100nm, such as about 82 to about 98nm, about 84 to about 96nm, about 86 to about 94nm, or about 88 to about 92nm. This particle size may produce a better performance in terms of immune cell uptake and immune response than other, larger particle sizes. The particle size may be stable (i.e. show no significant change), for instance when measured over a period of 1 month, 2 months, 3 months, 6 months, 12 months, 18 months, 24 months, 36 months or 48 months.

CaP nanoparticles can be co-formulated with one or multiple antigens either adsorbed on the surface of the nanoparticle or co-precipitated with CaP during particle synthesis. For example, a peptide, such as a hepatitis B virus peptide, may be attached to the CaP nanoparticle by dissolving the peptide in DMSO (for example at a concentration of about 10 mg/ml), adding to a suspension of CaP nanoparticles together with N-acetyl glucosamine (G1cNAc) (for example at 0.093mo1/L and ultra-pure water, and mixing at room temperature for a period of about 4 hours (for example, 1 hour, 2 hours, 3 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours or 10 hours).

The vaccine composition may comprise about 0.15 to about 0.8%, such as 0.2 to about 0.75%, 0.25 to about 0.7%, 0.3 to about 0.6%, 0.35 to about 0.65%, 0.4 to about 0.6%, or 0.45 to about 0.55%, CaP nanoparticles. Preferably the vaccine composition comprises about 0.3% CaP nanoparticles.

CaP nanoparticles have a high degree of biocompatibility due to their chemical similarity to human hard tissues such as bone and teeth. Advantageously, therefore, CaP nanoparticles are non-toxic when used for therapeutic applications. CaP nanoparticles are safe for administration via intramuscular, subcutaneous, oral, or inhalation routes. CaP nanoparticles are also simple to synthesise commercially. Furthermore, CaP nanoparticles may be associated with slow release of antigen, which may enhance the induction of an immune response to a peptide attached to the nanoparticle. CaP nanoparticles may be used both as an adjuvant, and as a drug delivery vehicle.

The nanoparticle may be a gold nanoparticle. Gold nanoparticles are known in the art and are described in particular in WO 2002/32404, WO 2006/037979, WO 2007/122388, WO 2007/015105 and WO 2013/034726. The gold nanoparticle attached to each peptide may be a gold nanoparticle described in any of WO 2002/32404, WO 2006/037979, WO 2007/122388, WO 2007/015105 and WO 2013/034726.

Gold nanoparticles comprise a core comprising a gold (Au) atom. The core may further comprise one or more Fe, Cu or Gd atoms. The core may be formed from a gold alloy, such as Au/Fe, Au/Cu, Au/Gd, Au/Fe/Cu, Au/Fe/Gd or Au/Fe/Cu/Gd. The total number of atoms in the core may be 100 to 500 atoms, such as 150 to 450, 200 to 400 or 250 to 350 atoms. The gold nanoparticle may have a mean diameter of 1 to 100, 20 to 90, 30 to 80, 40 to 70 or 50 to 60 nm. Preferably, the gold nanoparticle has a mean diameter of 20 to 40nm.

The nanoparticle may comprise a surface coated with alpha-galactose and/or betaGleNHAc. For instance, the nanoparticle may comprise a surface passivated with alpha-galactose and/or beta-GIcNHAc. In this case, the nanoparticle may, for example, be a nanoparticle which comprises a core including metal and/or semiconductor atoms. For instance, the nanoparticle may be a gold nanoparticle. Beta-G1cNHAc is a bacterial pathogen-associated-molecular pattern (PAW), which is capable of activating antigen-presenting cells. In this way, a nanoparticle comprising a surface coated or passivated with Beta-GleNHAc may non-specifically stimulate an immune response. Attachment of the hepatitis B virus peptide comprising one or more of the CDS+ T cell epitopes set out in SEQ ID NOs: 1 to 43 to such a nanoparticle may therefore improve the immune response elicited by administration of the vaccine composition of the invention to an individual. One or more ligands other than the peptide may be linked to the nanoparticle, which may be any of the types of nanoparticle described above. The ligands may form a "corona", a layer or coating which may partially or completely cover the surface of the core. The corona may be considered to be an organic layer that surrounds or partially surrounds the nanoparticle core. The corona may provide or participate in passivating the core of the nanoparticle. Thus, in certain cases the corona may be a sufficiently complete coating layer to stabilise the core. The corona may facilitate solubility, such as water solubility, of the nanoparticles of the present invention.

The nanoparticle may comprise at least 10, at least 20, at least 30, at least 40 or at least 50 ligands. The ligands may include one or more peptides, protein domains, nucleic acid molecules, lipidic groups, carbohydrate groups, anionic groups, or cationic groups, glycolipids and/or glycoproteins. The carbohydrate group may be a polysaccharide, an oligosaccharide or a monosaccharide group (e.g. glucose). One or more of the ligands may be a non-self component, that renders the nanoparticle more likely to be taken up by antigen presenting cells due to its similarity to a pathogenic component. For instance, one or more ligands may comprise a carbohydrate moiety (such as a bacterial carbohydrate moiety), a surfactant moiety and/or a glutathione moiety. Exemplary ligands include glucose, N-acetylglucosamine (G1cNAc), glutathione, 2'-thioethyl-Ii-D-glucopyranoside and 2'-thioethyl-D-glucopyranoside. Preferred ligands include glycoconjugates, which form glyconanoparticles Linkage of the ligands to the core may be facilitated by a linker. The linker may comprise a thiol group, an alkyl group, a glycol group or a peptide group. For instance, the linker may comprise C2-C15 alkyl and/or C2-C15 glycol. The linker may comprise a sulphur-containing group, amino-containing group, phosphate-containing group or oxygen-containing group that is capable of covalent attachment to the core. Alternatively, the ligands may be directly linked to the core, for example via a sulphur-containing group, amino-containing group, phosphate-containing group or oxygen-containing group comprised in the ligand.

Attachment to ncmoparticles The peptide may be attached at its N-terminus to the nanoparticle. Typically, the peptide is attached to the core of the nanoparticle, but attachment to the corona or a ligand may also be possible.

The peptide may be directly attached to the nanoparticle, for example by covalent bonding of an atom in a sulphur-containing group, amino-containing group, phosphate-containing group or oxygen-containing group in the peptide to an atom in the nanoparticle or its core.

A linker may be used to link the peptide to the nanoparticle. The linker may comprise a sulphur-containing group, amino-containing group, phosphate-containing group or oxygen-containing group that is capable of covalent attachment to an atom in the core. For example, the linker may comprise a thiol group, an alkyl group, a glycol group or a peptide group.

The linker may comprise a peptide portion and a non-peptide portion. The peptide portion may comprise the sequence X1X2Z1, wherein Xi is an amino acid selected from A and G; X7 is an amino acid selected from A and G; and Z1 is an amino acid selected from Y and F. The peptide portion may comprise the sequence AAY or FLAAY. The peptide portion of the linker may be linked to the N-terminus of the peptide. The non-peptide portion of the linker may comprise a C2-C15 alkyl and/ a C2-C15 glycol, for example a thioethyl group or a thiopropyl group.

The linker may be (i) HS-(CH2)2-CONH-AAY; (ii) HS-(C112)2-CONH-LAAY; (iii) HS-(CH2)3-CONH-AAY; (iv) HS-(CH2)3-CONH-FLAAY; (v) HS-(CH2)11)-(CH2OCH2)7-CONH-AAY; and (vi) HS-(CH2)i0-(CH2OCH2)7-CONH-FLAAY. In this case, the thiol group of the non-peptide portion of the linker links the linker to the core.

Other suitable linkers for attaching a peptide to a nanoparticle are known in the art, and may be readily identified and implemented by the skilled person.

As explained above, the vaccine composition may comprise multiple hepatitis B virus peptides each comprising one or more of the CD8+ T cell epitopes set out in SEQ ID NOs: 1 to 43 SEQ ID NOs: I to 43. The vaccine composition may comprise one or more additional peptides each comprising an epitope, such as a CD4+ T cell epitope, a B cell epitope, or a CD8+ T cell epitope other than the CD8+ T cell epitopes set out in SEQ ID NOs: I to 43 or SEQ ID NOs: 44 to 60. Thus, the vaccine composition may comprise more than one peptide.

When the vaccine composition comprises more than one peptide, two or more (such as three or more, four or more, five or more, ten or more, or twenty or more) of the peptides may be attached to the same nanoparticle. Two or more (such as three or more, four or more, five or more, ten or more, or twenty or more) of the peptides may each be attached to different nanoparticle. The nanoparticles to which the peptides are attached may though be the same type of nanoparticle. For instance, each peptide may be attached to a gold nanoparticle. Each peptide may be attached to a CaP nanoparticle. The nanoparticle to which the peptides are attached may be a different type of nanoparticle. For instance, one peptide may be attached to a gold nanoparticle, and another peptide may be attached to a CaP nanoparticle.

Medicaments, methods and therapeutic use The invention provides a method of preventing or treating a hepatitis B virus infection, comprising administering the vaccine composition of the invention to a subject infected with, or at risk of being infected with, a hepatitis B virus. The invention also provides a vaccine composition of the invention for use in a method of preventing or treating a hepatitis B virus infection in a subject; The hepatitis B virus infection may be, for example, a serotype adr, serotype adw, serotype ayr or serotype ayw hepatitis B virus infection. The hepatitis B virus infection 25 may be, for example, a genotype A, genotype B, genotype C, genotype D, genotype E, genotype F, genotype G, genotype H or genotype J hepatitis B virus infection.

The vaccine composition may be provided as a pharmaceutical composition. The pharmaceutical composition preferably comprises a pharmaceutically acceptable carrier or diluent. The pharmaceutical composition may be formulated using any suitable method.

Formulation of cells with standard pharmaceutically acceptable carriers and/or excipients may be carried out using routine methods in the pharmaceutical art. The exact nature of a formulation will depend upon several factors including the cells to be administered and the desired route of administration. Suitable types of formulation are fully described in Remington's Pharmaceutical Sciences, 19th Edition, Mack Publishing Company, Eastern Pennsylvania, USA.

The vaccine composition or pharmaceutical composition may be administered by any route. Suitable routes include, but are not limited to, the intravenous, intramuscular, intraperitoneal, subcutaneous, intradermal, transdermal and oral/buccal routes.

Compositions may be prepared together with a physiologically acceptable carrier or diluent. Typically, such compositions are prepared as liquid suspensions of peptides and/or peptide-linked nanoparticles. The peptides and/or peptide-linked nanoparticles may be mixed with an excipient which is pharmaceutically acceptable and compatible with the active ingredient. Suitable excipients are, for example, water, saline, dextrose, glycerol, of the like and combinations thereof.

In addition, if desired, the pharmaceutical compositions may contain minor amounts of auxiliary substances such as wetting or emulsifying agents, and/or pH buffering agents.

The peptides or peptide-linked nanoparticles are administered in a manner compatible with the dosage formulation and in such amount will be therapeutically effective. The quantity to be administered depends on the subject to be treated, the disease to be treated, and the capacity of the subject's immune system. Precise amounts of nanoparticles required to be administered may depend on the judgement of the practitioner and may be peculiar to each subject.

Any suitable number of peptides or peptide-linked nanoparticles may be administered to a subject. For example, at least, or about, 0.2 x 106, 0.25 x 106, 0.5 x 106, 1.5 x 106, 4.0 x 106 or 5.0 x 106 peptides or peptide-linked nanoparticles per kg of patient may administered. For example, at least, or about, 105, 106, 107, 10s, 109 peptides or peptide-linked nanoparticles may be administered. As a guide, the number of peptides or peptide-linked nanoparticles to be administered may be from 105 to 109, preferably from 106 to 10s.

It is to be understood that different applications of the disclosed products and methods may be tailored to the specific needs in the art. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments of the invention only, and is not intended to be limiting.

In addition, as used in this specification and the appended claims, the singular forms "a", "an", and "the" include plural referents unless the content clearly dictates otherwise. Thus, for example, reference to "a peptide" includes "peptides", reference to "a nanoparticle" includes two or more such nanoparticles, and the like.

All publications, patents and patent applications cited herein, whether supra or infra, are hereby incorporated by reference in their entirety.

The following Example illustrates the invention.

Example 1

The HLA-A2 and A24 positive liver hepatocellular carcinoma cell line HepG2 and its hepatitis B infected derivatives HepDE19 and HepG2.2.15 were cultured in Dulbecco's Modified Eagle Medium/Ham' s F-12 50/50 Mix (Mediatech Inc, Manassas VA). 293-T cells were maintained in Dulbecco's' Modified Eagle Medium and T2 cells were maintained in RPMI-1640 (Mediatech Inc). All medium was supplemented with 10% foetal bovine serum (Atlanta Biologicals, Flowery Branch, GA), L-glutamine (300mg/mL), lx non-essential amino acids, 0.5mM sodium pyruvate, and lx penicillin/streptomycin (Mediatech Inc). Cells were maintained at 37°C and 5% CO,.

Cell lysates were prepared from HBV infected cells and MHC/peptide complexes were isolated by immunoaffinity chromatography using MHC molecule specific antibodies (Testa el al. 2012). The peptides purified from the MHC molecules were fractionated using C-18 reversed phase (RP) column (4.6mm diameter x 150 mm length) using an offline HPLC (Dionex, Sunnyvale, CA). The peptide containing fractions were collected and dried to 6 pL under vacuum for LC/MS/MS analysis.

Mass spectrometry experiments were carried out using LTQ (Thermo) and Orbitrap 25 instruments interfaced with nano ultimate HPLC (Dionex). RP-HPLC purified peptide fractions were injected individually into the EC MS/NIS system to identify the sequences of the peptides. The peptides were analysed using a Data-Dependent method. The acquired spectra data were searched against all influenza strains protein database using Proteome Discoverer (Thermo) to interpret data and derive peptide sequences.

Synthetic peptides were made and subjected to LC-MS/MS analysis under identical experimental conditions as described above and their sequences were confirmed based on their MS/MS data. Candidate peptide sequences were confirmed by comparison of their MS/MS spectra with that of their synthetic analogs.

Forty-three MFIC class I-associated epitopes including HLA-A2 and A24 specific motifs were identified (Table 2). All the peptide sequences were present in multiple genotypes of HBV family.

Epitope HLA Parent Protein Accession ID SEQ ID binding NO: ADHGARLSL X protein [Hepatitis B virus] gi14211488 1 AICSVLRRA A2 polymerase [Hepatitis B virus] g 224978740 2 ALMPIYTCI A2/24 polymerase [Hepatitis B virus] gi452628 3 CFEHGEHHI A24 Polymerase [Hepatitis B virus] g 222824656 4 DLVPNMENI A2/24 middle S protein [Hepatitis B virus] g 209362964 5 DNQYGTNIQNLQN polymerase [Hepatitis B virus] g 112380514 6 ELGEENRLMIYVL A2/24 X protein [Hepatitis B virus] gi83314087 7 FDIRASSASS polymerase [Hepatitis B virus] g 190147544 8 FGATVELLTF HBcAb [Hepatitis B virus] gi306268 9 FLDPLLVLQA A2 HBsAg [Hepatitis B virus] gi58190162 10 GILTTVPTV A2 S protein [Hepatitis B virus] gi38198572 11 GRDLLDTARA precore/core ORF [Hepatitis B virus] gi9971629 12 GRPFSGPPGAL X peptide [Hepatitis B virus] g 198447151 13 GRPLPGPLGAVS Hbx protein [Hepatitis B virus] gi 108937192 14 GSPLSWEQELQ P protein [Hepatitis B virus] gi164416627 15 IFARIGDPA large S protein [Hepatitis B virus] gi209484443 16 ILLPMLIFLLV A2 middle S protein [Hepatitis B virus] gi2289202 17 ILTTMPAAPPSA A2 large surface protein [Hepatitis B virus] gi205362291 18 INNLANK SAS polymerase [Hepatitis B virus] g 11935073 19 ISSISSRTGDL S protein [Hepatitis B virus] gi38198448 20 ISTIPQSLDS surface antigen [Hepatitis B gi3328369 21 virus] LLDLKGMLP S protein [Hepatitis B virus] gi157411138 22 LLTKILII A2/24 S protein [Hepatitis B virus] gi205986003 23 LNSLVPFVQ S protein [Hepatitis B virus] gi 189171098 24 LYKWTLGL A24 X protein [Hepatitis B virus] gi27466468 25 LYNILSPFLL A24 S protein [Hepatitis B virus] gi157411152 26 METTVNAPLN X protein [Hepatitis B virus] gi193248379 27 MLASSNTSRGTC A3 S protein [Hepatitis B virus] gi162424181 28 NFLGGTIECL A24 S protein [Hepatitis B virus] gi225632851 29 NINYHHET A2 polymerase [Hepatitis B virus] gi112949722 30 PPASPPIVPS X peptide [Hepatitis B virus] gi6692552 31 QSKGPVLSCWLL polymerase [Hepatitis B virus] gi55274830 32 SSIYKRIGDP large surface protein [Hepatitis B virus] gi205362323 33 S SKPRKGMVT preS1/preS2 surface [Hepatitis B virus] gi51449942 34 TKPLLGNCTCIPI S protein [Hepatitis B virus] gi167507457 35 TLPNLHDNCSRNL polymerase [Hepatitis B virus] gi146746963 36 TVRKAADPA A2 polymerase [Hepatitis B virus] gi198076006 37 VLLGFAAPFTQ A24 polymerase [Hepatitis B virus] gi164654403 38 VTNLQSLTNLLS A2 polymerase [Hepatitis B virus] gi39748644 39 WVGSNLTFGREIV A24 core protein [Hepatitis B virus] g 226713196 40 WYLGPSLYSIVSL A24 S protein [Hepatitis B virus] gi164416610 41 YPGIQAIQA polymerase [Hepatitis B virus] gi224810371 42 YVDDVVLGAESV A2 reverse transcriptase [Hepatitis B virus] gi76251861 43 Table 2: List of identified HBV MFIC peptides, their sequences, corresponding proteins and accession IDs

Claims

CLAIMS1. A vaccine composition comprising a peptide comprising a CD8+ T cell epitope set out in any one of SEQ ID NOs: 1 to 43.
2. The vaccine composition of claim 1, wherein the peptide is 8 to 30 amino acids in length.
3 The vaccine composition of claim 1 or 2, wherein the CD8+ T cell epitope is capable of interacting with at least two different HLA supertypes.
4. The vaccine composition of claim 3, wherein the at least two different HLA supertypes are selected from HLA-Al, HLA-A2, HLA-A3, HLA-A24, HLA-B7, HLA-B8, HLA-B27, HLA-B44, HLA-B58 and HLA-B62.
5. The vaccine composition of claim 3, wherein the at least two different HLA supertypes are HLA-A2 and HLA-A24.
6. The vaccine composition of any one of the preceding claims, wherein the CD8+ T cell epitope is conserved between hepatitis B viruses.
7. The vaccine composition of any one of the preceding claims, wherein the peptide is attached to a nanoparticle.
8. The vaccine composition of claim 7, wherein the nanoparticle is a gold nanoparticle, a calcium phosphate nanoparticle, or a silicon nanoparticle.
9. The vaccine composition of claim 8, wherein the gold nanoparticle is coated with alpha-galactose and/or beta-G1cNHAc.
10. The vaccine composition of any one of claims 7 to 9, wherein the peptide is attached to the nanoparticle via a linker.
11. The vaccine composition of any one of the preceding claims, which comprises two or more hepatitis B peptides each comprising a different CD8+ T cell epitope.
12. The vaccine composition of claim 11, wherein each of the two or more peptides is: (a) a peptide as defined in any one of claims 1 to 6; or (b) a peptide comprising a CD8+ T cell epitope set out in any one of SEQ ID NOs: 44 to 60.
13. The vaccine composition of claim 11 or 12, wherein each of the two or more peptides is attached to a nanoparticle.
14. The vaccine composition of any one of claims 11 to 13, wherein each of the two or more peptides is capable of interacting with a different HLA supertype.
15. The vaccine composition of any one of the preceding claims, comprising a peptide comprising a CD4+ T cell epitope.
16. The vaccine composition of claim 15, wherein the CD4+ T cell epitopes interacts with all HLA class II types.
17. The vaccine composition of claim 15 or 16, wherein the CD4+ T cell epitope comprises the sequence set out in SEQ ID NO: 61 or 62.
18. A method of preventing or treating a hepatitis B virus infection, comprising administering the vaccine composition of any one of the preceding claims to an individual infected with, or at risk of being infected with, a hepatitis B virus.
19. The vaccine composition of any one of claims 1 to 17 for use in a method of preventing or treating a hepatitis B virus infection in an individual.
20. The method of claim 18 or vaccine composition for use of claim 19, wherein the hepatitis B virus infection is a serotype adr, serotype adw, serotype ayr or serotype ayw infection.
21. The method of claim 18 or vaccine composition for use of claim 19, wherein the hepatitis B virus infection is a genotype A, genotype B, genotype C, genotype D, genotype E, genotype F, genotype G, genotype H or genotype.1 infection.